Project Summary Nef, an HIV accessory protein critical for viral pathogenesis, prevents the adaptive immune system from detecting and destroying infected cells by downregulating MHC-I surface expression. Nef drives the mislocalization and degradation of MHC-I by recruiting the major clathrin adaptor protein, AP-1, to the Trans- Golgi Network and forcing AP-1:MHC-I complexes into clathrin coated vesicles destined for the lysosome. Structures of Nef in complex with AP-1 and its cofactor, Arf1, in the context of MHC-I binding revealed that AP- 1 trimerization and organization into a lattice could underpin cargo and MHC-I recognition, clathrin recruitment and subsequent vesicle formation. However, a major remaining gap in this ?lattice model? is that the interactions of AP-1, Nef, and MHC-I occur in, and are organized by, the lipid bilayer; all structures solved to date have been determined in the absence of a membrane. To fill this major void, the project uses powerful biochemical and structural tools to uncover the molecular mechanisms of AP-1 and Nef function in the context of membranes. In Aim 1, AP-1 cargo recognition at the membrane will be reconstituted in vitro and visualized by fluorescence microcopy in the presence and absence of Nef. AP-1:Arf1 is predicted to form large, punctate structures when recognizing endogenous cargo, and will specifically require Nef to form punctate structures when binding the non-cognate cargo, MHC-I. Clathrin is predicted to preferentially associate with punctate structures consistent with the hypothesis that AP-1 lattice formation underpins cargo recognition, Nef-dependent MHC-I trafficking and clathrin-mediated vesicle formation. To visualize the physical interactions that drive membrane binding, cargo recognition and cross-talk within the AP-1 complex, Aim 2 will solve the high-resolution structures of AP- 1:Arf1:Nef complexes bound to MHC-I cargo on nanodiscs by single particle cryo-EM. The near atomic-resolution structures will unveil critical missing structural information defining how contact with the membrane mediates Nef-dependent MHC-I recognition by AP-1. Aim 3 will bridge observations made from Aim 1 and Aim 2 by solving the medium-resolution structure of AP-1 complexes on a bona fide membrane using cryo-electron tomography. The sub-nanometer resolution electron density maps produced by subtomogram averaging will directly visualize the organization of AP-1 complexes on membranes for the first time and provide a template to build the first pseudo-atomic model of AP-1 cargo recognition and Nef hijacking activity. Strategic collaboration with the Collins and Kirchhoff labs will allow us to test the implications of our findings in vivo, thereby extending the impact directly to the HIV field. More broadly, the project establishes an in vitro platform for studying HIV accessory proteins and clathrin adaptors in a biologically relevant environment, e.g. on membranes, of which may be instrumental in uncovering mechanisms of HIV pathogenesis and/or screening compounds that combat Nef function or enable greater study of HIV biology.